Our long term goal is to leverage GPCR structures to discover new chemotypes, and use these to as leads and probes to disentangle signaling pathways. Libraries of over 4.4 Million commercially available molecules are docked against GPCRs, typically targeting allosteric sites, and those that rank well by a physics-based complementarity score are acquired for testing. Those that are confirmed are optimized for affinity, specificity, and permeability. We begin relatively conservatively, seeking ligands with new chemotypes and physical properties for the orthosteric site of muscarinic receptors, and build on this to target the allosteric sites newly revealed in the structures. The specific aims are: 1. Novel chemotypes for the orthosteric sites ofthe muscarinic receptors. We are particularly focused on chemotypes with new physical properties (e.g., uncharged ligands) and sub-type specificities. 2. Ligands to the Gs-binding site ofthe B2-AR. Combined with an orthosteric ligand, and potentially on their own, molecules that bind to this site will bias signaling down non-G-protein pathways, such as that of arrestin. 3. Allosteric ligands ofthe muscarinic receptor. The high sequence identity in the orthosteric site ofthe five muscarinic subtypes has interfered with the discovery of specific ligands. Sequence is much less conserved in the allosteric sites revealed in the new structures, and we are targeting these for sub-type specific ligands. 4. Dimer-site ligands for the u-opioid receptor. Molecules that bind to this site will stabilize dimer vs. momomer signaling, suggesting a new route to specificity among opioid receptors and new tools to investigate the role of oligomers in GPCR signaling. Whereas these goals are ambitious, extensive preliminary results, in collaboration with the Kobilka and the Sunahara labs, support their feasibility.